Enantiomer compositions of (1R,4S)-1-butyrylamino-4-(hydroxymethyl)-2-cyclopentene

ABSTRACT

A novel process for the preparation of (1S,4R)- or (1R,4S)-4-(2-amino-6-chloro-9H-purin-9-yl)-2-cyclopentene-1-methanol of the formulae ##STR1## is described. This entails in the first stage (±)-2-azabicyclo[2.2.1]hept-5-en-3-one of the formula ##STR2## being acylated to give a (±)-2-azabicyclo[2.2.1]hept-5-en-3-one derivative of the general formula ##STR3## in which R 1  denotes C 1-4  -alkyl, C 1-4  -alkoxy, aryl or aryloxy, the latter being reduced in the second stage to give a cyclopentene derivative of the general formula ##STR4## in which R 1  has the stated meaning, the latter then being converted in the third stage biotechnologically into the (1R,4S)- or (1S,4R)-1-amino-4-(hydroxymethyl)-2-cyclopentene of the formula ##STR5## the latter being converted in the fourth stage with N-(2-amino-4,6-dichloro-5-pyrimidinyl)formamide of the formula ##STR6## into the (1S,4R)- or (1R,4S)-4-[(2-amino-6-chloro-5-formamido-4-pyrimidinyl)amino]-2-cyclopentene-1-methanol of the formulae ##STR7## and the latter being cyclized in the fifth stage in a known manner to the final product of the formula I or II.

This is a divisional of pending U.S. application Ser. No. 09/073,553,filed May 6, 1998.

The invention relates to a novel process for the preparation of (1S,4R)-or (1R,4S)-4-(2-amino-6-chloro-9H-purin-9-yl)-2-cyclopentene-1-methanolof the formulae ##STR8## and to a novel process for preparing opticallyactive compounds of the general formulae ##STR9##

(1S,4R)-4-(2-Amino-6-chloro-9H-purin-9-yl)-2-cyclopentene-1-methanol isan important intermediate for preparing 2-aminopurine nucleosides suchas, for example, for preparing(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol(WO 95/21 161) or for preparing 1592U89 (J. Org. Chem., 1996, 61,4192-4193; J. Org. Chem., 1996, 61, 7963-7966).

A process for preparing(1S,4R)-4-(2-amino-6-chloro-9H-purin-9-yl)-2-cyclopentene-1-methanolstarting from (1S,4R)-4-amino-2-cyclopentene-1-methanol is described inWO 95/21 161. The disadvantage of this process is that the precursor(1S,4R)-4-amino-2-cyclopentene-1-methanol can be obtained only via(±)-2-azabicyclo[2.2.1]hept-5-en-3-one substituted by the costly BOCprotective group (tert-butyloxycarbonyl protective group) (J. Org.Chem., 1995, 60, 4602-4616).

The object of the invention was to provide a simple, low-cost and moreeconomical process for preparing (1S,4R)- or(1R,4S)-4-(2-amino-6-chloro-9H-purin-9-yl)-2-cyclopentene-1-methanol.

This object has been achieved by the novel process according to claim 1.

The first stage in the novel process is carried out by acylating(±)-2-azabicyclo[2.2.1]hept-5-en-3-one of the formula ##STR10## to givea (±)-2-azabicyclo[2.2.1]hept-5-en-3-one derivative of the generalformula ##STR11## in which R¹ denotes C₁₋₄ -alkyl, C₁₋₄ -alkoxy, aryl oraryloxy.

C₁₋₄ -Alkyl can be substituted or unsubstituted. Substituted C₁₋₄ -alkylmeans hereinafter C₁₋₄ -alkyl substituted by a halogen atom. F, Cl, Bror I can be used as halogen atom. Examples of C₁₋₄ -alkyl are methyl,ethyl, propyl, butyl, isobutyl, tert-butyl, isopropyl, chloromethyl,bromomethyl, dichloromethyl, dibromomethyl. Preferably used as C₁₋₄-alkyl is methyl, ethyl, propyl, butyl, isobutyl or chloromethyl.

It is possible to use as C₁₋₄ -alkoxy, for example, methoxy, ethoxy,propoxy, butoxy, tert-butoxy or isobutoxy. The C₁₋₄ -alkoxy preferablyused is tert-butoxy.

It is possible to use as aryl, for example, phenyl or benzyl, preferablyphenyl. Benzyloxy or phenoxy, for example, can be used as aryloxy.

The precursor (±)-2-azabicyclo[2.2.1]hept-5-en-3-one can be prepared asdisclosed in EP-A 0 508 352.

The acylation can be carried out with a carbonyl halide of the generalformula ##STR12## or with a carboxylic anhydride of the general formula##STR13## in which R¹ has the stated meaning, and X denotes a halogenatom. F, Cl, Br or I can be used as halogen atom. Cl or F is preferablyused.

Examples of carbonyl halides are: acetyl chloride, chloroacetylchloride, butyryl chloride, isobutyryl chloride, phenylacetyl chloride,benzyl chloroformate (Cbz-Cl), propionyl chloride, benzoyl chloride,allyl chloroformate or tert-butyloxycarbonyl fluoride. Examples ofcarboxylic anhydrides are: di-tert-butyl dicarbonate, butyric anhydride,acetic anhydride or propionic anhydride.

The acylation can be carried out without solvent or with an aproticsolvent.

The acylation is expediently carried out in an aprotic solvent. Examplesof suitable aprotic solvents are diisopropyl ether, pyridine,acetonitrile, dimethylformamide, triethylamine, tetrahydrofuran,toluene, ethylene chloride, N-methylpyrrolidone or mixtures hereof.

The acylation is expediently carried out at a temperature from -80 to50° C., preferably from 0 to 25° C.

In the second stage of the novel process, the(±)-2-azabicyclo[2.2.1]hept-5-en-3-one derivative of the formula IV isreduced to give a cyclopentene derivative of the general formula##STR14## in which R¹ has the stated meaning.

The reduction is expediently carried out with an alkali metalborohydride or alkaline earth metal borohydride, with an alkali metalaluminium hydride or alkaline earth metal aluminium hydride or withVitride (sodium bis(2-methoxyethoxy)aluminium hydride). Sodium orpotassium aluminium hydride can be used as alkali metal aluminiumhydride. Sodium or potassium borohydride can be used as alkali metalborohydride. Calcium borohydride can be used as alkaline earth metalborohydride. Aluminium nitride for example can be used as nitride.

The reduction is expediently carried out in a protic solvent. Proticsolvents which can be used are lower aliphatic alcohols such asmethanol, ethanol, propanol, isopropanol, butanol, sec-butanol,tertbutanol, isobutanol or water, or a mixture of the said alcohols andwater.

The reduction is expediently carried out at a temperature from -40 to40° C., preferably from 0 to 20° C.

The third stage in the novel process, the conversion of the cyclopentenederivative of the general formula V into the (1R,4S)- or(1S,4R)-1-amino-4-(hydroxymethyl)-2-cyclopentene of the formulae##STR15## is carried out either by means of microorganisms or of anenzyme having N-acetylamino-alcohol hydrolase activity or by means ofpenicillin G acylases. This biotransformation converts the acylated(1S,4R)- or (1R,4S)-amino alcohol derivative to result in the (1R,4S) or(1S,4R)-1-amino-4-(hydroxymethyl)-2-cyclopentene (formula VI, VII).

All microorganisms which utilize a cyclopentene derivative of thegeneral formula V as sole nitrogen source, as sole carbon source or assole carbon and nitrogen source are suitable for the biotransformation.The microorganisms can be isolated from soil samples, sludge or wastewater with the assistance of conventional microbiological techniques.The microorganisms are isolated by culturing them in a nutrient mediumcontaining a cyclopentene derivative of the general formula ##STR16## inwhich R¹ has the stated meaning, as sole carbon and nitrogen source

as sole nitrogen source with a suitable carbon source or

as sole carbon source with a suitable nitrogen source, in a conventionalway.

Examples of suitable cyclopentene derivatives of the general formula Vare: N-acetyl-, N-propionyl-, N-isobutyryl-,N-tert-butoxycarbonyl-(N-BOC), N-butyryl- orN-phenylacetyl-1-amino-4-hydroxymethyl-2-cyclopentene.

The microorganisms can use as suitable nitrogen source, for example,ammonium, nitrates, amino acids or ureas as substrate for growth. Themicroorganisms can use as suitable carbon source, for example, sugars,sugar alcohols, C₂ -C₄ -carboxylic acids or amino acids as substrate forgrowth. Hexoses such as glucose or pentoses can be used as sugars.Glycerol, for example, can be used as sugar alcohol. Acetic acid orpropionic acid can be used, for example, as C₂ -C₄ -carboxylic acids.Leucine, alanine, asparagine can be used, for example, as amino acids.

The selection medium and culture medium which can be used are thoseconventional among those skilled in the art, such as, for example, theone described in Table 1 or a complete medium (medium containing yeastextract) such as, for example, nutrient yeast broth (NYB), preferablyusing the one described in Table 1.

During the culturing and selection, the active enzymes of themicroorganisms are expediently induced. The cyclopentene derivatives ofthe general formula V can be used as enzyme inducer.

The culturing and selection normally takes place at a temperature from20° C. to 40° C., preferably from 30° C. to 38° C. and at a pH betweenpH 5.5 and pH 8, preferably between pH 6.8 and pH 7.8.

The biotransformation is expediently carried out with microorganismswhich utilize the (1R,4S) isomer of the cyclopentene derivative as solecarbon source, as sole carbon and nitrogen source or as sole nitrogensource.

The biotransformation is preferably carried out by means ofmicroorganisms of the genus Alcaligenes/Bordetella, Rhodococcus,Arthrobacter, Alcaligenes, Agrobacterium/Rhizobium, Bacillus,Pseudomonas or Gordona, in particular of the speciesAlcaligenes/Bordetella FB 188 (DSM 11172), Rhodococcus erythropolis CB101 (DSM 10686), Arthrobacter sp. HSZ5 (DSM 10328), Rhodococcus sp. FB387 (DSM 11291), Alcaligenes xylosoxydans ssp. denitrificans HSZ17 (DSM10329), Agrobacterium/Rhizobium HSZ30, Bacillus simplex K2, Pseudomonasputida K32 or Gordona sp. CB 100 (DSM 10687), and with theirfunctionally equivalent variants and mutants. Deposition in accordancewith the Budapest Treaty at the Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH, Mascheroderweg 1b, D38124 Braunschweig, tookplace on 20.05.1996 for the microorganisms DSM 10686 and 10687, on06.11.1995 for the microorganisms DSM 10328 and DSM 10329, on 08.10.1996for the microorganism DSM 11291 and on 20.09.1996 for the microorganismDSM 11172.

"Functionally equivalent variants and mutants" mean microorganismshaving essentially the same properties and functions as the originalmicroorganisms. Variants and mutants of this type can be produced bychance, for example by UV radiation.

Taxonomic description of Alcaligenes/Bordetella FB 188

    ______________________________________                                        (DSM 11172)                                                                   ______________________________________                                        Cell form          rods                                                         Width μm 0.5-0.6                                                           Length μm 1.0-2.5                                                          Motility +                                                                    Flagellation peritrichous                                                     Gram reaction -                                                               Lysis by 3% KOH +                                                             Aminopeptidase (Cerny) +                                                      Spores -                                                                      Oxidase +                                                                     Catalase +                                                                    ADH (alcohol dehydrogenase) -                                                 NO.sub.2 from NO.sub.3 -                                                      Denitrification -                                                             Urease -                                                                      Hydrolysis of gelatin -                                                       Acid from (OF test):                                                          Glucose -                                                                     Fructose -                                                                    Arabinose -                                                                   Adipate +                                                                     Caprate +                                                                     Citrate +                                                                     Malate +                                                                      Mannitol -                                                                  ______________________________________                                    

Taxonomic description of Rhodococcus erythropolis CB 101 (DSM 106 86)

1. Morphology and color of the colonies: short branched hyphae which,when old, disintegrate into rods and cocci, colonies glistening andpartly confluent, beige with pink tinge, RAL 1001;

2. Diagnosed amino acid of the peptidoglycan: mesodiaminopimelic acid;

3. Mycolic acids: Rhodococcus mycolic acids; determination of themycolic acid chain length (C₃₂ -C₄₄) and comparison of the data with theentries in the DSM mycolic acid data bank revealed very great similaritywith the patterns of the Rhodococcus erythropolis strains (similarity0.588).

4. Fatty acid pattern: unbranched, saturated and unsaturated fatty acidsplus tuberculostearic acid.

5. On partial sequencing of the 16S rDNA of the strain, a high level ofagreement (100%) was found with the sequences of the specific regions ofRhodococcus erythropolis.

The identification result is unambiguous because three mutuallyindependent methods (mycolic acids, fatty acids, 16S rDNA) have assignedthe strain to the species Rhodococcus erythropolis.

Taxonomic description of Gordona sp. CB 100 (DSM 10687)

1. Morphology and color of the colonies: short branched hyphae which,when old, disintegrate into rods and cocci, colonies pale orange, (RAL2008);

2. Diagnosed amino acid of the peptidoglycan: mesodiaminopimelic acid;

3. Menaquinone pattern: MK-9 (H₂) 100%;

4. Mycolic acids: Gordona mycolic acids; the mycolic acid chain length(C₅₀ -C₆₀) was determined by high temperature gas chromatography. Thispattern corresponds to the pattern found in representatives of the genusGordona.

5. Fatty acid pattern: unbranched, saturated and unsaturated fatty acidsplus tuberculostearic acid.

6. On partial sequencing of the 16S rDNA of the strain, only arelatively low agreement of 98.8% could be found with the sequences ofthe specific regions of Gordona rubropertincta.

On the basis of the available results (menaquinones, mycolic acids,fatty acids, 16S rDNA), although the isolate can be unambiguouslyassigned to the genus Gordona it is not possible on the basis of theresults to make an assignment to a known Gordona species. It istherefore to be assumed that the strain DSM 10687 is a new andpreviously undescribed species of the genus Gordona.

Taxonomic description of Alcaligenes xylosoxydans ssp. denitrificans HSZ17 (DSM 10329)

    ______________________________________                                        Properties of the strain                                                      ______________________________________                                        Cell form          rods                                                         Width μm 0.5-0.6                                                           Length μm 1.5-3.0                                                          Motility +                                                                    Flagellation peritrichous                                                     Gram reaction -                                                               Lysis by 3% KOH +                                                             Aminopeptidase (Cerny) +                                                      Spores -                                                                      Oxidase +                                                                     Catalase +                                                                    Anaerobic growth -                                                            ADH (alcohol dehydrogenase) +                                                 NO.sub.2 from NO.sub.3 +                                                      Denitrification +                                                             Urease -                                                                      Hydrolysis of                                                                 Gelatin -                                                                     Tween 80 -                                                                    Acid from (OF test):                                                          Glucose aerobic -                                                             Xylose 80 -                                                                   Substrate utilization                                                         Glucose -                                                                     Fructose -                                                                    Arabinose -                                                                   Citrate +                                                                     Malate +                                                                      Mannitol -                                                                  ______________________________________                                    

Taxonomic description of Arthrobacter sp. HSZ5 (DSM 10328)

    ______________________________________                                        Characterization: Gram-positive irregular                                        rods with a pronounced                                                        rod-cocci growth cycle;                                                       strictly aerobic; no acid                                                     or gas formation from                                                         glucose.                                                                     Motility -                                                                    Spores -                                                                      Catalase +                                                                  ______________________________________                                    

meso-Diaminopimelic acid in the cell wall: no Peptidoglkycan type: A3α,L-Lys-L-Ser-L-Thr-L-Ala 16S rDNA sequence similarity: The highest valuesfound on sequencing the region with the greatest variability were 98.2%with Arthrobacter pascens, A. ramosus and A. oxydans

Taxonomic description of Agrobacterium/Rhizobium HSZ30

    ______________________________________                                        Cell form         pleomorphic rods                                              Width [μm] 0.6-1.0                                                         Length [μm] 1.5-3.0                                                        Gram reaction -                                                               Lysis by 3% KOH +                                                             Aminopeptidase +                                                              Spores -                                                                      Oxidase +                                                                     Catalase +                                                                    Motility +                                                                    Anaerobic growth -                                                            Nitrite from nitrate -                                                        Denitrification -                                                             Urease +                                                                      Hydrolysis of gelatin -                                                       Acid from:                                                                    L-Arabinose +                                                                 Galactose -                                                                   Melezitose -                                                                  Fucose +                                                                      Arabitol -                                                                    Mannitol -                                                                    Erythritol -                                                                  Alkalinization of litmus milk +                                               Ketolactose -                                                               ______________________________________                                    

Partial sequencing of the 16S rDNA revealed comparably largesimilarities of about 96% with representatives of the generaAgrobacterium and Rhizobium. Unambiguous assignment to a speciesdescribed within these genera is not possible.

Taxonomic description of Bacillus simplex K2

    ______________________________________                                        Cell form          rods                                                         Width [μm] 0.8-1.0                                                         Length [μm] 3.0-5.0                                                        Spores -                                                                      Ellipsoidal -                                                                 Circular -                                                                    Sporangium -                                                                  Catalase +                                                                    Anaerobic growth -                                                            VP reaction n.g.                                                              Maximum temperature                                                           Growth positive at ° C. 40                                             Growth negative at ° C. 45                                             Growth in medium pH 5.7 -                                                     NaCl 2% +                                                                     5% -                                                                          7% -                                                                          10% -                                                                         Lysozyme medium +                                                             Acid from (ASS)                                                               D-Glucose +                                                                   L-Arabinose +                                                                 D-Xylose -                                                                    D-Mannitol +                                                                  D-Fructose +                                                                  Gas from fructose -                                                           Lecithinase -                                                                 Hydrolysis of                                                                 Starch +                                                                      Gelatin +                                                                     Casein -                                                                      Tween 80 +                                                                    Aesculin -                                                                    Utilization of                                                                Citrate +                                                                     Propionate -                                                                  Nitrite from nitrate +                                                        Indole -                                                                      Phenylalanine deaminase -                                                     Arginine dihydrolase -                                                      ______________________________________                                    

Analysis of the cellular fatty acids yielded confirmation of theassignment to the genus Bacillus.

Partial sequencing of the 16S rDNA revealed a similarity of 100% withBacillus simplex.

    ______________________________________                                        Taxonomic description of Pseudomonas putida K32                               ______________________________________                                        Cell form         rods                                                          Width [μm] 0.8-0.9                                                         Length [μm] 1.5-4.0                                                        Motility +                                                                    Flagellation polar >1                                                         Gram reaction -                                                               Lysis by 3% KOH +                                                             Aminopeptidase +                                                              Spores -                                                                      Oxidase +                                                                     Catalase +                                                                    Anaerobic growth -                                                            Pigments                                                                      fluorescent +                                                                 Pyocyanin -                                                                   ADH +                                                                         Nitrite from nitrate -                                                        Denitrification -                                                             Urease -                                                                      Hydrolysis of gelatin -                                                       Substrate utilization                                                         Adipate -                                                                     Citrate +                                                                     Malate +                                                                      D-Mandelate +                                                                 Phenylacetate +                                                               D-Tartrate -                                                                  D-Glucose +                                                                   Trehalose -                                                                   Mannitol -                                                                    Benzoylformate -                                                              Propylene glycol +                                                            Butylamine +                                                                  Benzylamine +                                                                 Tryptamine -                                                                  Acetamide +                                                                   Hippurate +                                                                 ______________________________________                                    

The profile of cellular fatty acids is typical of Pseudomonas putida.

Partial sequencing of the 16S rDNA revealed similarities of about 98%with Pseudomonas mendocina and Pseudomonas alcaligenes. The similaritywith Pseudomonas putida was 97.4%.

Taxonomic description of Rhodococcus sp. FB 387 (DSM 11291)

1. Morphology and colour of the colonies: short branched hyphae which,when old, disintegrate to rods and cocci, colonies matt, pale red-orangeRAL 2008;

2. Diagnosed amino acid of the peptidoglycan: mesodiaminopimelic acid;

3. Mycolic acids: Rhodococcus mycolic acids; Determination of themycolic acid chain length (C₃₂ -C₄₄) and comparison of the data with theentries in the DSMZ mycolic acid data bank revealed only very smallsimilarity with the patterns of Rhodococcus ruber strains (similarity0.019). This correlation factor is too low to be used for speciesidentification.

4. Fatty acid pattern: unbranched, saturated and unsaturated fatty acidsplus tuberculostearic acid. This fatty acid pattern is diagnostic forall representatives of the genus Rhodococcus and its close relativessuch as Mycobacterium, Nocardia and Gordona. An attempt was made byincluding the qualitative and quantitative differences in the fatty acidpattern to carry out a differentiation to the species level. Numericalmethods were used to compare the fatty acid pattern of Rhodococcus sp.FB 387 with the entries in the data bank. It was not possible with thismethod either to assign Rhodococcus sp. FB 387, because of the smallsimilarity (0.063), to any described Rhodococcus species.

5. On partial sequencing of the 16S rDNA of the strain, 96-818 wasassigned to Rhodococcus opacus with a correlation of 97.9%. Thissequence agreement is far below that of 99.5% required for unambiguousspecies assignment in this taxon.

On the basis of the available results, it can be assumed that the strainRhodococcus sp. FB 387 is a new and not previously described Rhodococcusspecies.

The biotransformation can be carried out, after conventional initialcultivation of these microorganisms, with quiescent cells (non-growingcells no longer requiring a carbon and energy source) or with growingcells. The biotransformation is preferably carried out with quiescentcells.

The enzymes with N-acetylamino-alcohol hydrolase activity which aresuitable for the biotransformation can be isolated from the describedmicroorganism cells for example by disruption as usual in the art. It ispossible to use for this for example the ultrasound or French pressmethod. These enzymes are preferably isolated from the microorganisms ofRhodococcus erythropolis CB101 (DSM 10686).

Suitable penicillin G acylases are obtained from many microorganismssuch as, for example, bacteria or actinomycetes, specifically from thefollowing micro-organisms: Escherichia coli ATCC 9637, Bacillusmegaterium, Streptomyces lavendulae ATCC 13664, Nocardia sp. ATCC 13635,Providencia rettgeri ATCC 9918, Arthrobacter viscosus ATCC 15294,Rhodococcus fascians ATCC 12975, Streptomyces phaeochromogenes ATCC21289, Achromobacter ATCC 23584 and Micrococcus roseus ATCC 416.Penicillin G acylases which can be bought are used in particular, suchas penicillin G acylase EC 3.5.1.11 from E.coli (Boehringer Mannheim) orfrom Bacillus megaterium.

Immobilized penicillin G acylases are used in a preferred embodiment.

The biotransformation can be carried out in media usual in the art, suchas, for example, in low-molarity phosphate, citrate or Hepes buffer, inwater, complete media such as, for example, Nutrient Yeast Broth (NYB)or in that described in the table. The biotransformation is preferablycarried out in the medium shown in Table 1 or in low-molarity phosphatebuffer.

The biotransformation is expediently carried out with a single orcontinuous addition of the cyclopentene derivative (formula V) so thatthe concentration does not exceed 10% by weight, preferably 2% byweight.

The pH during the biotransformation can be in a range from 5 to 9,preferably from 6 to 8. The biotransformation is expediently carried outat a temperature from 20 to 40° C., preferably from 25 to 30° C.

In the fourth stage, the cyclopentene derivative (formula VI or VII) isconverted with N-(2-amino-4,6-dichloro-5-pyrimidinyl)formamide of theformula ##STR17## into the (1S,4R)- or(1R,4S)-4-[(2-amino-6-chloro-5-formamido-4-pyrimidinyl)amino]-2-cyclopentene-1-methanolof the formula IX or X ##STR18##

The N-(2-amino-4,6-dichloro-5-pyrimidinyl)formamide can be prepared asdisclosed in WO 95/21 161.

The reaction in the fourth stage is expediently carried out in thepresence of a base. Organic bases or inorganic bases can be employed asbase. Trialkylamines can be used as organic bases. Examples oftrialkylamines used are triethylamine, tributylamine, tribenzylamine,pyridine or N-methylpyrrolidine. Examples of inorganic bases which canbe employed are alkali metal or alkaline earth metal carbonates oralkali metal or alkaline earth metal bicarbonates such as, for example,potassium carbonate and sodium bicarbonate.

The reaction in the fourth stage is expediently carried out in a proticsolvent. Lower aliphatic alcohols such as methanol, ethanol, propanol,isopropanol, butanol or isobutanol can be used as protic solvent.

The reaction in the fourth stage is expediently carried out at atemperature from 0 to 150° C., preferably from 20 to 100° C.

In the fifth stage, the (1S,4R)- or(1R,4S)-4-[(2-amino-6-chloro-5-formamido-4-pyrimidinyl)amino]-2-cyclopentene-1-methanol(formula IX, X) is cyclized in a known manner as disclosed in WO 95/21161 to give the final product of the formula I or II.

The cyclization is normally carried out dissolved in trialkylorthoformates in the presence of a concentrated aqueous acid. Examplesof trialkyl orthoformates which can be used are trimethyl or triethylorthoformate. It is possible to use as aqueous acid, for example,hydrochloric acid, sulphuric acid or methanesulphonic acid.

The invention further relates to a process for preparing the opticallyactive compounds of the general formulae ##STR19## in which R¹ has thestated meaning. These compounds can be obtained by converting acyclopentene derivative of the general formula ##STR20## in which R¹ hasthe stated meaning, by means of the microorganisms,N-acetylamino-alcohol hydrolases or penicillin G acylases which havealready been described previously into the (1R,4S)- or(1S,4R)-1-amino-4-(hydroxymethyl)-2-cyclopentene of the formulae##STR21## the latter being acylated to compounds of the formula XVI orXVII.

Both the microbiological conversion and the acylation are carried outunder conditions which are per se the same as described previously.

This acylation is expediently carried out at a temperature from 20° C.to 100° C., preferably from 0° C. to 80° C.

Examples of optically active compounds which are prepared by thisprocess are:(1R,4S)-N-tert-butoxycarbonyl-1-amino-4-(hydroxymethyl)-2-cyclopentenewith a ee of 98%,(1R,4S)-N-acetyl-1-amino-4-(hydroxymethyl)-2-cyclopentene with a ee of98%, (1R,4S)-N-butyryl-1-amino-4-(hydroxymethyl)-2-cyclopentene with aee of 98%, optically active(1S,4R)-N-acetyl-1-amino-4-(hydroxymethyl)-2-cyclopentene and opticallyactive (1S,4R)-N-butyryl-1-amino-4-(hydroxymethyl)-2-cyclopentene.

Of these compounds, the optically active(1R,4S)-N-butyryl-1-amino-4-(hydroxymethyl)-2-cyclopentene is a compoundwhich has not yet been described in the literature. Accordingly, theinvention also relates to optically active(1R,4S)-N-butyryl-1-amino-4-(hydroxymethyl)-2-cyclopentene with anenantiomeric excess of more than 0%, preferably in an enantiomericexcess of at least 80%, 90% or 95%, in particular in an enantiomericexcess of at least 98%. These optically active compounds can beracemized in a manner known to the skilled person to give racemicN-butyryl-1-amino-4-(hydroxymethyl)-2-cyclopentene which as not yet beendescribed in the literature.

The process for preparing a racemic or optically active4-(hydroxymethyl)-2-cyclopentene derivative of the general formula##STR22## in which R¹ has the stated meaning, is carried out by, in thefirst stage, acylating a cyclopentene-4-carboxylic acid of the formula##STR23## in the form of a racemate or one of its optically activeisomers, with a carbonyl halide of the general formula ##STR24## inwhich R¹ and X have the stated meaning, to give a racemic or opticallyactive cyclopentene-4-carboxylic acid derivative of the general formula##STR25## and, in the second stage, reducing the latter to the requiredproduct of the formula XIII.

Optically active 4-(hydroxymethyl)-2-cyclopentene derivatives (formulaXIII) and optically active cyclopentene-4-carboxylic acid derivatives(formula XV) mean the corresponding (1R,4S) or (1S,4R) isomers.

The first stage of this process, the acylation, is carried out with acarbonyl halide of the general formula XI. The carbonyl halides whichcan be used are the same as described previously, andtert-butyloxycarbonyl fluoride is preferably used.

The reaction in the first stage is expediently carried out at a pH from8 to 14, preferably from 12 to 14, and at a temperature from 0 to 50°C., preferably from 15 to 25° C.

Suitable solvents are mixtures of water with ethers. Ethers which can beused are dioxane, tetrahydrofuran, diethyl ether, glycol dimethyl ordiethyl ether.

The second stage of the process, the reduction, can be carried out withan alkali metal aluminium hydride, with a borane/di-C₁₋₄ -alkyl sulphidecomplex or with a borane/tetrahydrofuran complex. Lithium, sodium orpotassium aluminium hydride can be used as alkali metal aluminiumhydride, and lithium aluminium hydride is preferably used.Borane/dimethyl sulphide, borane/diethyl sulphide, borane/dipropylsulphide or borane/dibutyl sulphide complexes can be used asborane/di-C₁₋₄ -alkyl sulphide complex. Borane/dimethyl sulphide complexis preferably used.

It is expedient to use as solvent in the second stage one of the ethersmentioned above, without water.

The reaction in the second stage can be carried at a temperature from-50 to 5° C., preferably from -25 to -10° C.

EXAMPLES Example 1

Preparation of (±)-2-acetyl-2-azabicyclo[2.2.1]hept-5-en-3-one

100 g of (±)-2-azabicyclo[2.2.1]hept-5-en-3-one were dissolved inacetonitrile (800 ml) and pyridine (161.26 ml) under nitrogen. At 12°C., 104.5 g of acetyl chloride were added dropwise over the course of 2hours. The mixture was then stirred at room temperature for 4.5 hours.800 ml of water were added to the mixture, and the acetonitrile wasevaporated off in vacuo. The aqueous phase was extracted 3 times with400 ml of ethyl acetate. The combined org. phases were washed with 1NHCl (400 ml), water (400 ml), saturated NaCl (400 ml), dried withmagnesium sulphate and completely evaporated. The residue was taken upin methylene chloride and filtered through silica gel. The filtrate wasconcentrated and the product was purified by distillation. 107.76 g ofproduct were obtained as a clear liquid. The yield was 71%.

Boiling point (0.07 torr): 51° C.

    ______________________________________                                        .sup.1 H-NMR (CDCl.sub.3): δ [ppm]                                                           2.25 (AB syst., 2H);                                       400 MHz 2.8 (s, 3H);                                                           3.42 (m, 1H);                                                                 5.30 (m, 1H);                                                                 6.89 (m, 1H);                                                                 6.92 (m, 1H);                                                              ______________________________________                                    

Example 2

Preparation of (±)-2-butyryl-2-azabicyclo[2.2.1]hept-5-en-3-one

100.3 g of (±)-2-azabicyclo[2.2.1]hept-5-en-3-one were dissolved inacetonitrile (720 ml) and pyridine (142 ml) under nitrogen. At 12° C.,141.8 g of butyryl chloride were added dropwise over the course of 1hour. The reaction was then stirred at room temperature for 3 hours. 720ml of water were added to the mixture, and the phases were separated.The acetonitrile was evaporated off in vacuo, and the aqueous phase wasextracted 3 times with ethyl acetate (300 ml). The combined org. phaseswere washed with 1N HCl (350 ml), saturated NaCl (400 ml) and water (500ml), dried with magnesium sulphate and completely evaporated. Theproduct was purified by distillation. 107.76 g of product were obtainedas a clear liquid. The yield was 85%.

Boiling point (0.05 torr): 70° C.

    ______________________________________                                        .sup.1 H-NMR (CDCl.sub.3): δ [ppm]                                                         0.98 (t, J = 8.5 Hz, 3H);                                    400 MHz 1.58-1.65 (2H);                                                        2.23 (AB syst., 2H);                                                          2.82-2.90 (2H);                                                               3.42 (m, 1H);                                                                 5.30 (m, 1H);                                                                 6.62 (m, 1H);                                                                 6.90 (m, 1H).                                                              ______________________________________                                    

Example 3

Preparation of (±)-2-phenylacetyl-2-azabicyclo[2.2.1]-hept-5-en-3-one

33.4 g of (±)-2-azabicyclo[2.2.1]hept-5-en-3-one were dissolved inacetonitrile (240 ml) and pyridine (48.3 ml) under nitrogen. At 12° C.,68.6 g of phenylacetyl chloride were added dropwise over the course of30 minutes. The mixture was then stirred at room temperature for 3.5hours. 240 ml of water were added to the mixture. The acetonitrile wasevaporated off in vacuo, and the aqueous phase was extracted 3 timeswith ethyl acetate (150 ml). The combined org. phases were washed with1N HCl (150 ml), saturated NaCl (150 ml) and water (150 ml), dried withmagnesium sulphate and completely evaporated. The crude product wasfiltered through silica gel (hexane:ethyl acetate=1:1). 78.34 g of thecrude product were obtained as a yellow oil.

Example 4

Preparation of (±)-2-propionyl-2-azabicyclo[2.2.1]hept-5-en-3-one

47 g of (±)-2-azabicyclo[2.2.1]hept-5-en-3-one were dissolved inacetonitrile (325 ml) and pyridine (41 ml) under nitrogen. At 12° C.,43.9 g of propionyl chloride were added dropwise over the course of 1 h.The reaction was then stirred at room temperature for 5 h. 145 ml ofwater were added to the mixture, and the acetonitrile was evaporated offin vacuo. The aqueous phase was extracted 3 times with 115 ml of ethylacetate. The combined organic phases were washed with 1N HCl (140 ml),saturated NaHCO₃ (40 ml) and NaCl (40 ml) solutions, dried with sodiumsulphate and completely evaporated. The residue was purified bydistillation. 55.8 g of title compound were obtained and solidified onleaving to stand. The yield was 81.6%.

Boiling point 2.8 mbar 75-80° C. Melting point: 54-56° C.

    ______________________________________                                        .sup.1 H-NMR (DMSO-d.sub.6): δ [ppm]                                                          0.95 (t, 3H);                                             400 MHz 2.10 (quart., 1H);                                                     2.28 (quart., 1H);                                                            2.64 (m, 2H);                                                                 3.42 (s, 1H);                                                                 5.16 (s, 1H);                                                                 6.78 (m, 1H);                                                                 6.96 (m, 1H).                                                              ______________________________________                                    

Example 5

Preparation of (±)-2-isobutyryl-2-azabicyclo[2.2.1]hept-5-en-3-one

45.1 g of (±)-2-azabicyclo[2.2.1]hept-5-en-3-one were dissolved inacetonitrile (310 ml) and pyridine (39 ml) under nitrogen. At 10° C.,54.1 g of isobutyryl chloride were added dropwise over the course of 1h. The reaction was then stirred at room temperature for 5 h. 140 ml ofwater were added to the mixture, and the acetonitrile was evaporated offin vacuo. The aqueous phase was extracted with 4×120 ml of ethylacetate. The combined organic phases were washed with 1N HCl (50 ml),saturated NaHCO₃ (50 ml) and NaCl (50 ml) solutions, dried with sodiumsulphate and completely evaporated. The residue was boiled under refluxin n-hexane (240 ml) with active charcoal. After filtration of theactive charcoal, the filtrate was cooled to 0° C. and the title compoundwas filtered. 54.5 g of product were obtained. The yield was 76%.

Melting point: 41-42° C.

    ______________________________________                                        .sup.1 H-NMR (DMSO-d.sub.6): δ [ppm]                                                           0.92 (d, 3H);                                            400 MHz 1.06 (d, 3H);                                                          2.10 (m, 1H);                                                                 2.28 (m, 1H);                                                                 3.40 (m, 2H);                                                                 5.16 (s, 1H);                                                                 6.76 (m, 1H);                                                                 7.92 (m, 1H).                                                              ______________________________________                                    

Example 6

Preparation of (±)-2-chloroacetyl-2-azabicyclo[2.2.1]-hept-5-en-3-one

10.1 g of (±)-2-azabicyclo[2.2.1]hept-5-en-3-one were dissolved in amixture of dichloromethane (10 ml), pyridine (8.4 ml) and 0.22 g of4-N,N-dimethylaminopyridine at 10° C. under nitrogen. 13.5 g ofchloroacetyl chloride were added dropwise over the course of 1 h. Thetemperature rose to 44° C. The mixture was stirred for a further 2 h atroom temperature. 100 ml of water were added to the solution. Afterphase separation, the aqueous phase was extracted with 100 ml ofdichloromethane. The combined organic phases were dried with sodiumsulphate and completely evaporated. The residue was boiled in 100 ml ofdiisopropyl ether under reflux in the presence of 1 g of active charcoalfor 10 minutes. After hot filtration, the filtrate was cooled to roomtemperature, and the solid was filtered and dried. 10.35 g of titlecompound were obtained. The yield was 60%.

Melting point: 86-88° C.

    ______________________________________                                        .sup.1 H-NMR (CDCl.sub.3): δ [ppm]                                                             2.28 (d, 1R)                                             400 MHz 2.40 (d, 1H);                                                          3.48 (s, 1H);                                                                 4.56 (d, 2H);                                                                 5.30 (s, 1H);                                                                 6.70 (d, 1H);                                                                 6.94 (m, 1H).                                                              ______________________________________                                    

Example 7

Preparation of (±)-1-acetylamino-4-(hydroxymethyl)-2-cyclopentene

79.56 g of (±)-2-acetyl-2-azabicyclo[2.2.1]hept-5-en-3-one weredissolved in ethanol (450 ml) under nitrogen and cooled to -10° C. 19.8g of sodium borohydride were added in portions over the course of 45minutes.

The reaction was stirred at 0° C. for 3 hours and then the pH wasadjusted to 1.8 with conc. sulphuric acid. Ethyl acetate (200 ml) wasadded to this mixture, and the solids were filtered off. It was thencompletely evaporated. The residue was taken up in water, washed withmethylene chloride and evaporated. The crude product was purified by asilica gel filtration. 51.83 g of product were obtained as a whitesolid. The yield was 64% based on2-acetyl-2-azabicyclo[2.2.1]hept-5-en-3-one employed.

    ______________________________________                                        .sup.1 H-NMR (DMSO-d.sub.6): δ [ppm]                                                           1.18 (m, 1H);                                            400 MHz 1.78 (s, 3H);                                                          2.29 (m, 1H);                                                                 2.66 (m, 1H);                                                                 3.35 (s, 2H);                                                                 4.58 (s, 1H);                                                                 4.72 (m, 1H);                                                                 5.61 (d, 1H);                                                                 5.65 (d, 1H);                                                                 7.83 (d, 1H).                                                              ______________________________________                                    

Example 8

Preparation of (±)-1-butyrylamino-4-(hydroxymethyl)-2-cyclopentene

73.87 g of (±)-2-butyryl-2-azabicyclo[2.2.1]hept-5-en-3-one weredissolved in ethanol (400 ml) under nitrogen and cooled to -10° C. 15.68g of sodium borohydride were added in portions over the course of 45minutes. The reaction was stirred at 0° C. for 3 hours and then the pHwas adjusted to 1.5 with conc. sulphuric acid. Ethyl acetate (200 ml)was added to this mixture, and the solids were filtered off. It was thencompletely evaporated. The residue was taken up in water, washed withmethylene chloride, evaporated and dried under high vacuum. 60.55 g ofproduct were obtained. The yield was 80% based on(±)-2-butyryl-2-azabicyclo[2.2.1]hept-5-en-3-one employed.

Melting point: 71-72° C.

    ______________________________________                                        .sup.1 H-NMR (CDCl.sub.3): δ [ppm]                                                         0.98 (t, J = 8.5 Hz, 3H);                                    400 MHz 1.40-1.50 (1H);                                                        1.58-1.68 (2H);                                                               2.10-2.18 (2H);                                                               2.42-2.55 (1H);                                                               2.85 (m, 1H);                                                                 3.62 (AB syst., 2H);                                                          4.98 (m, 1H);                                                                 5.78-5.82 (2H);                                                               6.38 (m, 1H).                                                              ______________________________________                                    

Example 9

Preparation of (±)-1-phenylacetylamino-4-(hydroxymethyl)-2-cyclopentene

77 g of crude (±)-2-phenylacetyl-2-azabicyclo[2.2.1]hept-5-en-3-one weredissolved in ethanol (450 ml) under nitrogen and cooled to -10° C. 13.2g of sodium borohydride were added in portions over the course of 1hour. The reaction was stirred at room temperature for 3.5 hours andthen the pH was adjusted to 1.8 with conc. sulphuric acid. This mixturewas purified by a silica gel filtration (hexane:ethyl acetate=2:8).After recrystallization from ethyl acetate, 15.89 g of white solid wereobtained. The yield was 80% based on(±)-2-phenylacetyl-2-azabicyclo[2.2.1]hept-5-en-3-one employed.

    ______________________________________                                        .sup.1 H-NMR (CDCl.sub.3): δ [ppm]                                                           1.28-1.35 (1H);                                            400 MHz 1.40 (m, 1H);                                                          2.38-2.45 (1H);                                                               2.79 (m, 1H);                                                                 3.50 (AB syst., 2H);                                                          3.52 (s, 3H);                                                                 4.98 (m, 1H);                                                                 5.75 (m, 2H);                                                                 5.98 (m, 1H).                                                                 7.20-7.38 (5H).                                                            ______________________________________                                    

Example 10

Preparation of (±)-1-BOC-amino-4-(hydroxymethyl)-2-cyclopentene(BOC=tert-butoxycarbonyl)

15 g of crude (±)-1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride(prepared as described in J. Org. Chem. 1981, 46, 3268) were dissolvedin a mixture of 150 ml of water and 150 ml of dioxane at roomtemperature under nitrogen. The solution was adjusted to pH 14 with 1NNaOH, then a diethyl ether solution of tert-butyloxycarbonyl fluoride(BOC-F, 20% excess) was added, and the mixture was stirred for a further3 h at room temperature (BOC-F prepared as disclosed in Synthesis 1975,599). The pH was adjusted to 2 with conc. HCl . After distillation ofthe organic solvents, 50 ml of water were added to the residue, and themixture was extracted with 3×100 ml of ethyl acetate. The combinedorganic phases were completely evaporated. The residue was crystallizedin a mixture of 110 ml of diisopropyl ether and 80 ml of n-hexane. 11.95g of title compound were obtained. The yield was 56%.

Melting point: 68-70° C.

    ______________________________________                                        .sup.1 H-NMR (DMSO-d.sub.6): δ [ppm]                                                           1.18 (m, 1H);                                            400 MHz 1.38 (s, 9H);                                                          2.26 (m, 1H);                                                                 2.65 (m, 1H);                                                                 3.33 (t, 2H);                                                                 4.45 (m, 1H);                                                                 4.55 (t, 1H);                                                                 5.62 (m, 1H);                                                                 5.79 (m, 1H);                                                                 6.73 (d, 1H).                                                              ______________________________________                                    

Example 11

Preparation of (±)-1-propionylamino-4-(hydroxymethyl)-2-cyclopentene

16.6 g of (±)-2-propionyl-2-azabicyclo[2.2.1-hept-5-en-3-one weredissolved in water (140 ml) and 2-butanol (66 ml) under nitrogen andcooled to -5° C. 3 g of sodium borohydride were added in portions overthe course of 2 h. The mixture was stirred at 10° C. for 2.5 h and thenadjusted to pH 2.2 with a mixture of conc. hydrochloric acid and water(1/1) The solution was evaporated to 40 g and adjusted to pH 6.2 with 2NNaOH. The mixture was extracted with 5×50 ml of dichloromethane. Thecombine d organic phases were completely evaporated, and the residue wasrecrystallized in toluene (150 ml). 11.1 g of title compound wereobtained. The yield was 65%.

Melting point: 67-68° C.

    ______________________________________                                        .sup.1 H-NMR (DMSO-d.sub.6): δ [ppm]                                                          0.96 (t, 3H);                                             400 MHz 1.16 (quint., 1H);                                                     2.04 (quart., 2H);                                                            2.26 (m, 1H);                                                                 2.66 (m, 1H);                                                                 3.34 (m, 2H);                                                                 4.58 (t, 1H);                                                                 4.72 (m, 1H);                                                                 5.61 (m, 1H),                                                                 5.84 (m, 1H),                                                                 7.72 (d, 1H).                                                              ______________________________________                                    

Example 12

Preparation of (±)-1-isobutyrylamino-4-(hydroxymethyl)-2-cyclopentene

9 g of (±)-2-isobutyryl-2-azabicyclo[2.2.1]hept-5-en-3-one weredissolved in water (32 ml) and 2-butanol (84 ml) under nitrogen andcooled to 0° C. 1.37 g of sodium borohydride were added in portions overthe course of 3.5 h. The mixture was stirred for a further 3 h at 20°C., and it was then adjusted to pH 2.5 with a mixture of conc.hydrochloric acid and water (1/1) and then neutralized with 2N NaOH. Thesolution was evaporated to 40 g. The residue was extracted with 3×80 mlof dichloromethane. The combined organic phases were completelyevaporated. The resulting solid was crystallized in 25 ml of toluene.6.8 g of title compound were obtained. The yield was 73.6%.

Melting point: 80-81° C.

    ______________________________________                                        .sup.1 H-NMR (DMSO-d.sub.6): δ [ppm]                                                          0.98 (d, 6H);                                             400 MHz 1.16 (quint., 1H);                                                     2.30 (m, 2H);                                                                 2.68 (m, 1H);                                                                 3.32 (t, 2H);                                                                 4.58 (t, 1H);                                                                 4.70 (m, 1H);                                                                 5.61 (m, 1H);                                                                 5.82 (m, 1H);                                                                 7.68 (d, 1H).                                                              ______________________________________                                    

Example 13

Preparation of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene usingpenicillin G acylases

Penicillin G acylase EC 3.5.1.11 from E.coli (Boehringer Mannheim) 165 U(units)/g or penicillin G acylase EC 3.5.1.11 from Bacillus megateriumwas employed for the biotransformation.

For this, 50 mM sodium phosphate buffer (pH 5-9; 4 ml) was incubatedwith 1% by weight of non-racemic1-phenylacetylamino-4-hydroxymethyl-2-cyclopentene and 400 mg of theappropriate penicillin G acylase at 37° C.

Samples were taken after defined time intervals and were analyzed bythin-layer chromatography (silica gel 60, butanol:water:glacial aceticacid=3:1:1; detection with ninhydrin) gas chromatography (capillarycolumn, HP-5, 5% phenylmethylsiloxane) or HPLC. The enzyme eliminatedthe phenylacetyl group with high activity and thereby liberated up to40% of the corresponding amino alcohol. The free amino alcohol wasobtained with a ee of 80%.

Example 14

Preparation of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene usingmicroorganisms

14.1 Sewage sludge (20%) from the ARA water treatment plant in Visp wasincubated in the A+N medium (see Table 1) containing 0.5% by weight of1-acetyl-, 1-propionyl-, 1-isobutyryl- or1-butyrylamino-4-hydroxymethyl-2-cyclopentene at 37° C. with shaking.The formation of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene wasfollowed by thin-layer chromatography. 1-3 transfers were carried outwith 1% of these enrichments, and isolation took place on solid media(plate count agar in the medium of Table 1; 20 g/l). The microorganismsAlcaligenes/Bordetella FB 188 (DSM 1172), Rhodococcus erythropolis CB101 (DSM 10686), Gordona sp. CB 100 (DSM 10687) and Rhodococcus sp. FB387 (DSM 11291) were isolated in this way.

14.2 The microorganisms isolated in this way were cultivated in themedium (Table 1) containing 0.5% of 1-acetyl-, 1-propionyl-,1-isobutyryl- or 1-butyrylamino-4-hydroxymethyl-2-cyclopentene. Theygrew to an optical density (OD) of 2 to 3 in 24 to 36 hours. The cellsobtained in this way were harvested in the late exponential phase ofgrowth and were washed in 10 mM phosphate buffer.

The subsequent biotransformation was carried out in 50 mM phosphatebuffer (pH 4.5-9) containing 1% by weight of 1-acetyl-, 1-isobutyryl- or1-butyrylamino-4-hydroxymethyl-2-cyclopentene. It was found bythin-layer chromatography that 50% of the substrate were hydrolyzed to(1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene. HPLC analyses revealedee values between 80 and 93%.

When 1-butyrylamino-4-hydroxymethyl-2-cyclopentene was employed assubstrate, the biotransformation rate was 0.14 (g/l/h/OD) for the strainDSM 10686

when cultivation took place on a A+N medium and 0.03 (g/l/h/OD) whencultivation took place on NYB (nutrient yeast broth) medium containing1-butyrylamino-4-hydroxymethyl-2-cyclopentene.

When the same conversion was carried out with the strain DSM 10687 at asubstrate concentration (1-butyrylamino-4-hydroxymethyl-2-cyclopentene)of 200 mM, the biotransformation rate was 0.161 (g/l/h/OD).

                  TABLE 1                                                         ______________________________________                                        A + N medium                                                                  ______________________________________                                        MgCl.sub.2            0.4    g/l                                                CaCl.sub.2 0.014 g/l                                                          FeCl.sub.3 0.8 mg/l                                                           Na.sub.2 SO.sub.4 0.1 g/l                                                     KH.sub.2 PO.sub.4 1 g/l                                                       Na.sub.2 HPO.sub.4 2.5 g/l                                                    NaCl 3 g/l                                                                    Vitamin solution 1 ml/l                                                       Trace element solution 1 m/l                                                  pH 7.5                                                                      ______________________________________                                    

14.3 Rhodococcus erythropolis DSM 10686 was cultured in minimal medium(cf. Table 2) with ammonium acetate (3 g/l) as carbon and nitrogensource in a 6 l fermenter at 30° C. to a cell density of OD 650>25.During cell growth, 50% acetic acid was added continuously as additionalC source. In order to induce the enzymatic activity, 60 g of(+/-)-1-acetylamino-4-hydroxymethyl-2-cyclopentene were then added, andincubation was continued for some hours. Finally, a further 40 g of(+/-)-1-acetylamino-4-hydroxymethyl-2-cyclopentene were added, andincubation was then carried out for a further 10 hours. The progress ofthe biotransformation was followed on-line by HPLC. is When ananalytical yield of 40%, based on the racemic substrate employed, and aee of 85% were reached, fermentation was stopped by adding acid.

                  TABLE 2                                                         ______________________________________                                        Media composition                                                                   Component           Concentration                                       ______________________________________                                        Yeast extract         0.5    g/l                                                Peptone M66 0.5 g/l                                                           KH.sub.2 PO.sub.4 4.0 g/l                                                     Na.sub.2 HPO.sub.4 2H.sub.2 O 0.5 g/l                                         K.sub.2 SO.sub.4 2.0 g/l                                                      NH.sub.4 acetate 3.0 g/l                                                      CaCl.sub.2 0.2 g/l                                                            MgCl.sub.2.6H.sub.2 O 1.0 g/l                                                 Trace element solution 1.5 ml/l                                               (see below)                                                                   PPG (polypropylene glycol) 0.1 g/l                                            Trace element solution                                                        KOH 15.1 g/l                                                                  EDTA.Na.sub.2.2H.sub.2 O 100.0 g/l                                            ZnSO.sub.4.7H.sub.2 O 9.0 g/l                                                 MnCl.sub.2.4H.sub.2 O 4.0 g/l                                                 H.sub.3 BO.sub.3 2.7 g/l                                                      CoCl.sub.2.6H.sub.2 O 1.8 g/l                                                 CuCl.sub.2.2H.sub.2 O 1.5 g/l                                                 NiCl.sub.2.6H.sub.2 O 0.18 g/l                                                Na.sub.2 MoO.sub.4.2H.sub.2 O 0.27 g/l                                      ______________________________________                                    

14.4 In analogy to Example 14.3, the microorganisms Arthrobacter sp. HSZ5 (DSM 10328, Rhodococcus sp. FB387 (DSM 11291), Alcaligenesxylosoxydans ssp. denitrificans HSZ 17 (DSM 10329),Agrobacterium/Rhizobium HSZ 30, Bacillus simplex K2 and Pseudomonasputida K32 were cultured on sodium acetate in the medium (Table 1) withand without 1-acetyl-, 1-propionyl-, 1-isobutyryl- or1-butyrylamino-4-hydroxymethyl-2-cyclopentene, abbreviated to aminoalcohols hereinafter.

The following results were obtained with exponential cells culturedwithout amino alcohols (HPLC analysis):

    ______________________________________                                        Strain                                                                          [%] Rate [mmol/OD.h] ee/conversion                                          ______________________________________                                        HSZ 5 (DSM 10328)                                                                            0.05         88.7/16                                             HSZ 17 (DSM 10329) 0.005 95/23                                                K32 0.05 54/1                                                                 CB101 (DSM 10686) 0.1 84/39                                                 ______________________________________                                    

The strains K2 and K17 were cultured, harvested and subjected to a60-hour biotransformation.

    ______________________________________                                        Strain                                                                          [%] Rate [mmol/OD.h] ee/conversion                                          ______________________________________                                        K2         --           92/10                                                   HSZ 30 --  93/3.5                                                           ______________________________________                                    

Exponential and stationary cells were harvested from all the batches andemployed as quiescent cells for the biotransformation. There was noobservable difference, from the TLC analysis, in the initial rate ofcells induced with amino alcohol or not induced.

Example 15

Purification of the N-acetylamino-alcohol hydrolase from Rhodococcuserythropolis CB101 (DSM 10686)

The enzyme was purified as described below until there was only oneprotein band in the SDS-PAGE (Pharmacia Phast gel, 10-15% gradient) at amolecular weight of 50 kD.

Cells of Rhodococcus erythropolis CB101 (DSM 10686) were washed in 50 mMtris buffer (pH 6.2) and concentrated to an optical density OD₆₅₀ nm of190. After addition of phenylmethanesulphonyl fluoride (PMSF) to a finalconcentration of 1 mM and DNAse, the cells were treated with a Frenchpress in order to obtain a crude extract. Centrifugation resulted in 200ml of a cell-free extract with a protein concentration of 4.8 mg ml⁻¹.

960 mg of the cell-free extract were loaded onto a HiLoad 26/10Q-Sepharose ion exchange chromatography column (Pharmacia) which hadbeen equilibrated with a 50 mM tris buffer (pH 8.0) containing 1 mMdithiothreitol (DTT).

After the column had been washed with the same buffer, the proteins wereeluted with a linear buffer gradient (1500 ml; gradient: 50 mM trisbuffer (pH 8.0) containing 1 mM DTT--50 mM tris buffer (pH 7.0)containing 1 mM DTT and 1 M NaCl). The enzyme eluted from the columnbetween 370 and 430 mM NaCl and at a pH of 7.6. The active fractionswere collected and concentrated to 9 ml. The protein content was 41 mg.

For further purification, the protein solution was loaded onto a HiLoad26/60 Superdex 75 gel filtration chromatography column (Pharmacia) whichhad been equilibrated with a 50 mM tris buffer containing 50 mM NaCl and1 mM DTT. The active fractions were combined and had a total proteincontent of 10.9 mg.

This protein solution was loaded onto a Mono Q HR5/5 column (Pharmacia)which had been equilibrated with 50 mM tris buffer (pH 8.5) containing 1mM DTT. The proteins were eluted with a linear gradient (40 ml) of 50 mMtris buffer (pH 8.5) containing 1 mM DTT--50 mM tris buffer (pH 8.5)containing 1 mM DTT and 1 M NaCl. The enzyme eluted between 390 mM NaCland 440 mM NaCl. The active fractions contained 1.4 mg of protein.

In the last purification step, the same column was used, equilibratedwith the same buffer. The elution gradient used was the same buffer with0-500 mM NaCl and pH 7.0-8.5. It was possible in this way to isolate 430μg of pure enzyme.

The N-terminal sequence of the enzyme was determined directly from theprotein blot. A sequence of the following 20 amino acids was obtained:Thr-Glu-Gln-Asn-Leu-His-Trp-Leu-Ser-Ala-Thr-Glu-Met-Ala-Ala-Ser-Val-Ala-Ser-Asn.

This sequence showed no homology with known proteins.

Example 16

Enzyme Characterization

The enzyme characterization was carried out both with purified enzymeand with cell-free extract which had been desalted using a Sephadex G-25column (PD-10, Pharmacia).

The protein concentration in the cell-free extract was 7.3 mg ml⁻¹ andthe protein concentration of the purified enzyme was 135 μg ml⁻¹. PMSFwas not added to the cell-free extract.

16.1 K_(m) determination

The K_(m) determination was carried out in a cell-free extract. TheK_(m) for the reaction at pH 7.0 and at a temperature of 30° C. was 22.5mM for the substrate 1-acetylamino-4-hydroxymethyl-2-cyclopentene.

16.2 pH optimum

The pH optimum for the hydrolysis of1-acetylamino-4-hydroxymethyl-2-cyclopentene (25 mM) was determined withthe purified enzyme and in cell-free extract in a pH range of pH 6.2-9.0in the following buffer solutions.

Tris buffer 100 mM pH 9.0; 8.5; 8.0; 7.5; 7.0 Citrate/phosphate buffer100 mM pH 7.0; 6.55; 6.2

The activity was measured for 24 h.

The pH optimum for the reaction was between pH 7.0 and pH 7.5 forproduction of the 1R,4S and the 1S,4R enantiomer.

The pH optimum for the activity in the cell-free extract was at pH 7.0.The selectivity was, however, better between pH 6.0 and pH 7.0.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the activity of the N-acetylaminoalcohol hydrolase(cell-free extract) from Rhodococcus erythropolis CB 101 (DSM 10686) asa function of the pH. The temperature optimum for the reaction indicatedin Example 16.2 was between 25 and 30° C.

FIG. 2 shows the activity of the N-acetylaminoalcohol hydrolase(cell-free extract) from Rhodococcus erythropolis CB 101 (DSM 10686) asa function of the temperature.

16.4 The molecular weight was determined by SDS-PAGE to be 50 kD.

16.5 The following substrates were hydrolyzed:1-acetyl-amino-4-hydroxymethyl-2-cyclopentene,1-butyryl-amino-4-hydroxymethyl-2-cyclopentene,1-propionyl-amino-4-hydroxymethyl-2-cyclopentene,1-isobutyryl-amino-4-hydroxymethyl-2-cyclopentene.

Example 17

Preparation of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopenteneHydrochloride

374.1 g of a solution of (1R,4S)-1-amino-4-hydroxymethyl-2-cyclopentene(preparation similar to that in Example 14) were evaporated to 123.7 g.The solution contained 60.2 mmol of the above compound (HPLC) and wasadjusted from pH 2 to pH 11.7 with 30% strength NaOH and then extractedwith 3×70 ml of isobutanol. The combined isobutanol extracts wereadjusted to pH 1 with gaseous HCl, concentrated to 65 g and filtered(removal of solid impurities). 60 ml of acetone were added dropwise tothe vigorously stirred filtrate at 20° C. The cloudy mixture was seededwith crystals of the title compound and stirred at 5° C. for 1 h.Filtration and drying resulted in 5.2 g of product. The yield was 58%.

Melting point: 125-127° C. ee 98% (calibrated chiral HPLC column)

    ______________________________________                                        .sup.1 H-NMR (DMSO-d.sub.6): δ [ppm]                                                           1.44 (m, 1H);                                            400 MHz 2.35 (m, 1H);                                                          2.83 (m, 1H);                                                                 3.42 (m, 2H);                                                                 4.10 (s, 1H);                                                                 4.80 (s, 1H);                                                                 5.80 (d, 1H);                                                                 6.06 (d, 1H);                                                                 8.13 (s, 3H).                                                              ______________________________________                                    

Example 18

Preparation of (1R,4S)-1-BOC-amino-4-(hydroxymethyl)-2-cyclopentene

75 g of a solution of (1R,4S)-1-amino-4-hydroxymethyl-2-cyclopentene(preparation similar to that in Example 14; 44.6 mmol of the compounds)were adjusted to pH 8 with 30% strength NaOH, and 6 g of NaHCO₃ wereadded. The mixture was heated to 52° C. While stirring vigorously, 60 mlof diisopropyl ether were added and then a solution of 11.12 g of BOCanhydride in 18.2 ml of diisopropyl ether was metered in over the courseof 2 h. The mixture was filtered through Celite, and the phases wereseparated. The aqueous phase was extracted with 65 ml of diisopropylether. The combined organic phases were washed with 45 ml of water andthen evaporated to 37.5 g and heated to 50° C. 31 ml of n-hexane wereadded dropwise to the solution. After slowly cooling to 0° C. (2 h), thetitle compound was filtered, washed with 12 ml of n-hexane/diisopropylether 1/1 and dried. 6.75 g of product were obtained. The yield was 71%.

Melting point: 70-71° C. ee 98% (calibrated chiral HPLC column)

    ______________________________________                                        .sup.1 H-NMR (DMSO-d.sub.6): δ [ppm]                                                           1.18 (m, 1H);                                            400 MHz 1.27 (s, 9H);                                                          2.28 (m, 1H);                                                                 2.63 (m, 1H);                                                                 3.33 (q, 2H);                                                                 4.43 (m, 1H);                                                                 4.56 (t, 1H);                                                                 5.62 (m, 1H);                                                                 5.79 (m, 1H);                                                                 6.72 (d, 1H).                                                              ______________________________________                                    

Example 19

Preparation of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopenteneHydrochloride

87.8 g of (1R,4S)-1-BOC-amino-4-hydroxymethyl-2-cyclopentene weredissolved in 270 ml of 2N HCl and 1340 ml of methanol. The mixture wasboiled under reflux for 4.5 h. After distillation of the methanol, theresidue was dissolved in 800 ml of water. The aqueous solution wasextracted twice with 340 ml of ethyl acetate. The aqueous phase wascompletely evaporated (50° C./60 mbar). The solid was dried at 50° C. invacuo, suspended in 150 ml of diethyl ether, filtered off and washedtwice with 50 ml of diethyl ether. Drying resulted in the title compoundin a yield of 95% (58.4 g).

The physical and spectroscopic data of the product were the same as inExample 17.

Example 20

Preparation of (1R,4S)-1-acetylamino-4-(hydroxymethyl)-2-cyclopentene

25 g of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene hydrochloridewere dissolved in 182 ml of acetic anhydride and, at 0° C., a solutionof 18.25 g of triethylamine in 60 ml of acetic anhydride was added. Themixture was stirred at 80° C. for 3 h and then cooled to roomtemperature. The triethylamine hydrochloride was filtered off and washedwith 120 ml of n-hexane. The filtrate was evaporated. The residue wasmixed with 300 ml of toluene and stirred at room temperature in thepresence of 5.2 g of active charcoal and 13 g of Celite for 20 min.After filtration and washing of the filter cake (3×40 ml of toluene),the solvent was completely evaporated off. The residue was mixed with180 ml of methanol and 15.5 g of K₂ CO₃ and stirred at room temperaturefor 10 h. The suspension was filtered and the filtrate was evaporated.The residue was suspended in 750 ml of isopropyl acetate and boiled inthe presence of 0.5 g of active charcoal under reflux for 1.5 h. Afterthe active charcoal filtration (70-80° C.), the filtrate was cooled at0° C. overnight. The title compound was filtered, washed with 80 ml ofcold isopropyl acetate and dried in vacuo. 17.2 g of product wereobtained. The yield was 66%.

Melting point: 77-80° C. ee 98% (calibrated chiral HPLC column)

    ______________________________________                                        .sup.1 H-NMR (DMSO-d.sub.6): δ [ppm]                                                           1.15 (m, 1H);                                            400 MHz 1.78 (s, 3H);                                                          2.25 (m, 1H);                                                                 2.66 (m, 1H);                                                                 3.35 (m, 2H);                                                                 4.58 (t, 1H);                                                                 4.70 (m, 1H);                                                                 5.62 (m, 1H);                                                                 5.85 (m, 1H);                                                                 7.80 (d, 1H).                                                              ______________________________________                                    

Example 21

Preparation of (1S,4R)-1-acetylamino-4-(hydroxymethyl)-2-cyclopentene

It was possible to prepare the title enantiomer by the process ofExample 18 starting from 25 g of(1S,4R)-1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride (yield68%). The spectroscopic and physical data of the product were the sameas in Example 20.

Example 22

Preparation of (1R,4S)-1-butyrylamino-4-(hydroxymethyl)-2-cyclopentene

34.7 g of (1R,4S)-1-amino-4-hydroxymethyl-2-cyclopentene hydrochlorideand 2 g of 4-N,N-dimethylaminopyridine were dissolved in 600 ml ofmethylene chloride. The solution was cooled to 0° C. Then 52 g oftriethylamine were added dropwise (5 min). The mixture was stirred for afurther 30 min. A solution of 35.2 g of butyryl chloride in 60 ml ofmethylene chloride was metered into the mixture at 0C over the course of1 h. The mixture was stirred for a further 1.5 h at between 0 and 20°C., and then 600 ml of water were added. After phase separation, theaqueous phase was extracted with 600 ml of methylene chloride. Thecombined organic phases were washed 3 times with 500 ml of 10% strengthNaOH each time, and then completely evaporated. The dried solid wasdissolved in 120 ml of methanol. The solution was mixed with 5 g of K₂CO₃ and stirred for a further 2 h at room temperature. The inorganicsalts were filtered off and washed with 20 ml of methanol. The filtratewas neutralized with 2N HCl. The suspension was filtered and the filtercake was washed with 20 ml of methanol. The filtrate was completelyevaporated. The solid residue was dried and crystallized in 150 ml oftoluene. 28.5 g of title compound were obtained. The yield was 67%.

Melting point: 71-72° C. ee 98% (calibrated chiral HPLC column)

    ______________________________________                                        .sup.1 H-NMR (DMSO-d.sub.6): δ [ppm]                                                           0.85 (t, 3H);                                            400 MHz 1.15 (m, 1H);                                                          1.50 (q, 2H);                                                                 2.03 (d, 2H);                                                                 2.28 (m, 1H);                                                                 2.67 (m, 1H);                                                                 3.35 (d, 2H);                                                                 4.62 (s, 1H);                                                                 4.76 (m, 1H);                                                                 5.63 (m, 1H);                                                                 5.85 (m, 1H);                                                                 7.77 (d, 1H).                                                              ______________________________________                                    

Example 23

Preparation of (1S,4R)-1-butyrylamino-4-(hydroxymethyl)-2-cyclopentene

It was possible to prepare the title enantiomer by the method of Example20 starting from 34.7 g of(1S,4R)-1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride (yield63%). The spectroscopic and physical data of the product were the sameas in Example 22.

Example 24

Preparation of(1R,4S)-1-[(2-amino-6-chloro-5-formamido-4-pyrimidinyl)amino]-4-(hydroxymethyl)-2-cyclopentene

2.07 g of N-(2-amino-4,6-dichloro-5-pyrimidinyl)formamide were heated to80° C. in 40 ml of isobutanol (white suspension). A solution of 1.97 gof (1R,4S)-1-amino-4-hydroxymethyl-2-cyclopentene hydrochloride, 3.8 gof triethylamine and 15 ml of isobutanol was added to the mixture. Themixture was stirred at 80° C. for a further 13 h. 10 ml of 1N NaOH wereadded to the clear solution at 20° C., followed by evaporation todryness. The residue underwent flash chromatography (silica gel 60column, length 8 cm, diameter 6.5 cm, eluent ethyl acetate/methanol95/5). After evaporating off the eluent and drying the residue, 2.1 g oftitle compound were obtained. The yield was 74%.

Melting point: 174-176° C. ee 98% (calibrated chiral HPLC columns

    ______________________________________                                        .sup.1 H-NMR (DMSO-d.sub.6): δ [ppm]                                                        1.37 (m, 1H);                                               400 MHz 2.35 (m, 1H);                                                          2.73 (m, 1H);                                                                 3.38 (t, 2H),                                                                 4.68 (m, 1H);                                                                 5.08 (m, 1H);                                                                 5.70 (d, 1H);                                                                 5.85 (d, 1H);                                                                 6.40; 6.55 and 6.65 (s,d                                                     and d, together 3H);                                                           7.78 and 8.10 (d and s,                                                       together 1H);                                                                 8.55 and 8.95 (d and s,                                                       together 1H).                                                              ______________________________________                                    

Example 25

Preparation of(1R,4S)-1-[(2-amino-6-chloro-5-formamido-4-pyrimidinyl)amino]-4-(hydroxymethyl)-2-cyclopentene

145.2 ml of a solution of (1R,4S)-1-amino-4-hydroxymethyl-2-cyclopentene(preparation similar to that in Example 14) were concentrated to 25.5 mland filtered through Celite. The filter cake was washed with 7.5 ml ofwater. The filtrate contained 17.7 mmol of the above compound (HPLC) andwas adjusted from pH 6.6 to pH 1 with conc. HCl and then extracted 3times with 20 ml of isobutanol. The organic phase was discarded. Theaqueous phase was adjusted to pH 12 with 30% strength NaOH and extracted3 times with 10 ml of isobutanol. The combined organic phases wereevaporated to 15 ml and 2.53 g of triethylamine were added. A solutionof 2.07 g of N-(2-amino-4,6-dichloro-5-pyrimidinyl)formamide in 40 ml ofethanol was added to the mixture as in Example 24. The mixture wasstirred at 80° C. for 16 h. Working up as in Example 22 resulted in 2.4g of title compound. The yield was 85%.

The physical and spectroscopic data of the product were the same as inExample 24.

Example 26

Preparation of (±)-1-BOC-amino-2-cyclopentene-4-carboxylic Acid

16.4 g of crude (±)-1-amino-2-cyclopentene-4-carboxylic acidhydrochloride (prepared as in J. Org. Chem. 1981, 46, 3268) weredissolved in a mixture of 80 ml of water and 80 ml of dioxane at roomtemperature under nitrogen. The mixture was then adjusted to pH 14 with1N NaOH, and a diethyl ether solution of tert-butyloxycarbonyl fluoride(BOC-F, 20% excess) was 5 added (BOC-F prepared as in Synthesis 1975,599). The mixture was stirred for a further 5 h at room temperature. ThepH was adjusted to 2 with conc. HCl. After distillation of the organicsolvents, 50 ml of water were added to the residue, and the mixture wasextracted 3 times with 100 ml of ethyl acetate. The organic phase wasevaporated to 50 ml and diluted with 25 ml of toluene. After cooling(0-10° C.), the title compound was filtered and dried. 14.3 g of productwere obtained. The yield was 63%.

Melting point: 126-127° C.

    ______________________________________                                        .sup.1 H-NMR (DMSO-d.sub.6): δ [ppm]                                                           1.14 (s, 9H);                                            400 MHz 1.70 (m, 1H);                                                          2.40 (m, 1H);                                                                 3.40 (m, 1H);                                                                 4.47 (m, 1H);                                                                 5.70 (t, 1H);                                                                 4.87 (t, 1H);                                                                 6.88 (d, 1H);                                                                 12.30 (d, 1H).                                                             ______________________________________                                    

Example 27

Preparation of (±)-1-BOC-amino-4-(hydroxymethyl)-2-cyclopentene from(±)-1-BOC-amino-2-cyclopentene-4-carboxylic Acid

250 ml of tetrahydrofuran and 10.02 g (0.164 mol) of LiAlH₄ wereintroduced into a 500 ml stirring apparatus, and a solution of 30.0 g(0.132 mol) of (±)-1-BOC-amino-2-cyclopentene-4-carboxylic acid in 75 mlof tetrahydrofuran was metered in at -10° C. over the course of 1 h.Then 10 g of water, 10 g of 15% strength NaOH solution and 20 g of waterwere added, followed by filtration. The residue was washed twice with100 ml of tert-butyl methyl ether each time, and the combined organicphases were evaporated to dryness. After addition of 80 ml of hexane andseeding with the compound of Example 10, the title compound separatedout as crystalline solid. Yield 15.84 g (56%). The physical andspectroscopic data were the same as in Example 10.

Example 28

Preparation of (1R,4S)-1-BOC-amino-4-(hydroxymethyl)-2-cyclopentene from(1R,4S)-1-BOC-amino-2-cyclopentene-4-carboxylic Acid

80 ml of tetrahydrofuran and 11.38 g (50.07 mmol) of(1R,4S)-1-BOC-amino-2-cyclopentene-4-carboxylic acid (prepared as inTetrahedron: Asymmetry 1993, 4, 1117) were introduced into a 500 mlstirring apparatus, and 5 ml of borane/dimethyl sulphide complex weremetered in at -15° C. over the course of 1 h, and the mixture was thenstirred at this temperature for 3 h. A solution of 4 g of NaOH in 60 mlof water was added and warmed to room temperature. Extraction withtoluene, filtration through silica gel and subsequent crystallization inethyl acetate/n-hexane 1:1 afforded 5.4 g of the title compound,corresponding to a yield of 57%, as a white crystalline solid. Thephysical and spectroscopic data were the same as in Example 10. The eewas 99% (same chiral HPLC column).

What is claimed is:
 1. The compound consisting of (1R, 4S) or (4R,1S)-1-butyrylamino-4-(hydroxymethyl)-2-cyclopenetene of the formula##STR26## with an enantiomeric excess of more than 0%.
 2. The Enantiomeraccording to claim 1, in an enantiomeric excess of at least 80%.
 3. TheEnantiomer according to claim 1, in an enantiomeric excess of at least90%.
 4. The Enantiomer according to claim 1, in an enantiomeric excessof at least 95%.
 5. The Enantiomer according to claim 1, in anenantiomeric excess of at least 98%.
 6. A racemic compound of (1R, 4S)1-butyrylamino-4-(hydroxymethyl)-2-cyclopentene and (4R, 1S)1-butyrlamino-4-(hydroxymethyl)-2-cyclopentene.